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Clenbuterol hydrochloride magnetic particle separation enzyme-linked immunosorbent assay method

A technology of clenbuterol hydrochloride and magnetic particles, applied in the direction of color/spectral property measurement, measurement device, analysis material, etc., can solve the problems of weakened bronchodilation, increased concentration, endocrine disorder, etc., and achieves good repeatability, The effect of high sensitivity and simple processing method

Inactive Publication Date: 2010-12-08
北京倍爱康生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Long-term intake of sub-therapeutic doses, the gradual accumulation of hormone residues and chronic toxicity can cause hyposensitivity in the human body, that is, the bronchodilator effect is significantly weakened, and the duration of the effect is shortened, which increases the incidence and severity of asthma. At the same time, it can also cause endocrine Disorders that lead to increased concentrations of lactic acid and pyruvate in the blood, which may lead to acidosis in diabetics

Method used

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  • Clenbuterol hydrochloride magnetic particle separation enzyme-linked immunosorbent assay method
  • Clenbuterol hydrochloride magnetic particle separation enzyme-linked immunosorbent assay method

Examples

Experimental program
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Effect test

Embodiment 1

[0027] Example 1: Anti-FITC antigen coupled with surface amino (COOH-) magnetic particles to prepare magnetic separation reagent

[0028] Take 100 mg of magnetic particles containing carboxyl (COOH-) active groups on the surface and wash them three times with 0.1 M MES (2-[N-morpholino] ethanesulfonic acid), 10 ml of pH 4.5-5 solution. The magnetic particles were resuspended in 1ml of this solution, and 2mg of anti-FITC antibody was added, and mixed evenly. Add 100 μl of 10 mg / ml EDC solution, mix well and react at room temperature for 2 hours. After washing the magnetic beads 3 times with 10 ml of 0.01 M phosphate buffered saline (PBS) pH 7.4 containing 1% bovine serum albumin (BSA), the solution was used to prepare a 2.5 mg / ml magnetic separation reagent working solution.

Embodiment 2

[0029] Example 2: CLB-BSA linked alkaline phosphatase (ALP), preparation of enzyme-labeled antigen reagent

[0030] Take 5 mg of CLB-BSA antigen, concentrate to 5 mg / ml, add 10 μl of 13.76 mg / ml activator 2-Iminothiolane HCl (2IT) solution, place at room temperature for 30 minutes, add glycine to terminate the activation reaction, and place at room temperature for 5 minutes. Use Sephadex G25 column to desalt and collect protein elution peaks.

[0031] Take 5 mg of ALP solution, add 50 μl of 6.69 mg / ml Succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) solution, leave it at room temperature for 15 minutes, add glycine to terminate the activation reaction, and leave it at room temperature for 5 minutes. Use Sephadex G25 column to desalt and collect protein elution peaks.

[0032] The activated CLB-BSA was mixed with the activated ALP, left at room temperature for 10 hours, then separated and purified using Supperdex200 gel chromatography column to remove unlinke...

Embodiment 3

[0035] Example 3: Anti-CLB antibody linked to FITC to prepare anti-reagent

[0036] Take 5mg of anti-CLB antibody solution and dialyze against 20mM pH9.0 carbonate buffer for more than 12h, and the concentration after dialysis is required to be greater than 1mg / ml. Add 500 μl of 0.5 mg / ml FITC solution (prepared with 20 mM pH9.0 carbonic acid buffer), mix well and react at room temperature for more than 12 hours. Use Sephadex G25 column to remove unbound FITC, and collect the protein elution peak.

[0037] The preparation method of the anti-reagent diluent is the same as that of the enzyme-labeled antigen reagent diluent. The above-mentioned CLB antibody FITC conjugate is diluted to 1-5 μg / ml with the diluent to prepare the anti-reagent working solution.

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Abstract

The invention provides a clenbuterol (CLB) magnetic particle separation enzyme-linked immunosorbent assay (ELISA) method, belonging to the technical field of food safety immunodetection. The method comprises the following steps: the immunodetection principle of the competition method is adopted to connect CLB with bio-enzyme and prepare an enzyme labeled antigen reagent, antibody against fluorescein isothiocyanate (FITC) is absorbed on the surface of magnetic particles to prepare a magnetic separation reagent, FITC is connected with the antibody against CLB to prepare an antibody reagent; CLB in a sample and enzyme-labeled CLB compete to combine with a small quantity of FITC-labeled antibody against CLB and form an antigen-antibody composite; after the magnetic separation reagent is added, the antibody against FITC connected with the surface of magnetic particles captures the composite on the surface of magnetic particles; and washing, and finally adding substrate to detect. The invention has the following advantages: (1) magnetic particles replace the traditional ELISA plate to be used as solid carrier and ensure that the immunoreaction is performed more fully and fast under the near-liquidus condition; compared with the traditional ELISA method, the method is characterized by high specificity and good repeatability; and (2) the one-step competition method principle is adopted, thus the detection time is short.

Description

technical field [0001] The invention belongs to the technical field of immune detection and analysis, relates to food safety-related immunological detection and analysis technology, and in particular provides a magnetic particle separation enzyme-linked immunological detection method for rapidly detecting Clenbuterol hydrochloride (CLB), which is suitable for urine , quantitative detection of clenbuterol in liver, meat and other tissues. Background technique [0002] Clenbuterol (CLB) is an antiasthmatic drug. The drug is neither a veterinary drug nor a feed additive, but an adrenal nerve stimulant. Clenbuterol is a β2-receptor agonist. In the early 1980s, a company in the United States began to add it to feed to increase lean meat percentage. However, if it is used as a feed additive, the dosage is 10 times that of human medicine. Above, in order to achieve the effect of increasing the lean meat rate. It is used in a large amount, used for a long time, and metabolized sl...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/558G01N33/577G01N21/31
Inventor 韩学栋韩子华郭健夫仝文斌
Owner 北京倍爱康生物技术有限公司
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